Sarcoma antigen



Patented July 11, 1939 I SABCOMA ANTIGEN Benjamin Gruskin,Philadelphia,Pa., assignor to Lakeland Foundation, Chicago, 111., acorporation of Illinois No Drawing. Application October 6, 1936, SerialNo. 104,342

9 Claims.

-More specifically the invention is concerned.

with the diagnosis of malignant tumors and comprehends the. provision ofan extract of a material comprising a characteristic protein which willincite a specific sensitization to a homologous protein contained in thebody of an animal by producing a pseudopodic reaction when introducedintradermally into the said animal body. In view of this action thepresent agent or antigen may also or alternatively be termed asensitizing agent. By the term antigen as used in the followingspecification and claims it is intended to cover and refer to suchproducts.

More particularly, the invention contemplates the provision of anantigen for use in the diagnosis of malignant tumors known as cancer,there being two types of malignant tumors, namely, carcinoma andsarcoma. Carcinoma is a malignant tumor of the epithelial (cells formingan unbroken sheet or membrane) type, while the connective tissue type isknown as sarcoma. This invention and discovery relates to the antigenfor use in connection with the diagnosis of the connective tissue typeof tumor, which is sarcoma.

This application is a continuation in part of application Serial Nos.636,023 and 636,024, each 3 filed October 3, '1932, and of applicationSerial No. 731,115, filed June 18, 1934.

The present invention is based upon and explained by the followingtheory which is set out herein for the purpose of better defining theinvention and "the principles involved therein, namely, that malignanttumor cells are embryonic in their inception and continue to remainembryonic, in contra-distinction to other cells of the'body which areembryonic in inception but which later mature. That is to say, theembryon'ic connective tissue cells of sarcoma never mature but tend toremain in their embryonic form, thus characterizing the onset of thedisease. It is a biological fact that if an extract of a knownhomologous protein, which is a substance characteristic of livingmatter, is added to or brought into contact with serum of an animal thathas been injected with that protein, a precipitation or flocculation, ora reaction or immunization takes place. conventionally availed andemployed in the iden; tificatlon of various types of proteinaceousanimal products,'for example, by inspection agencies. Therefore, uponthe theory herein expounded,

and of which innumerable tests-have proven This biological fact iscorrect, an antigenmade of embryonic connective tissue cells added to orbrought into contact -with the serum of a patient sufiering fromsarcoma, will cause a reaction or immunization, or flocculation or'precipitation to take place. So also, when an antigen made fromembryonic connective tissue cells and containing a specific proteinhomologous to the protein characteristic of sarcoma is intradermallyinjected into the body of a patient suffering from sarcoma acharacteristic allergic or pseudopodic reaction occurs.

The primary object of the present invention is to provide a new antigenwhich may be used in determining whether or not a person is afflictedwith sarcoma.

Another object is the provision of an antigen for use in the diagnosisof sarcoma to determine whether or not malignancy exists by the scienceof serum diagnosis or serologically testing the serum of a patient.

A further object is the provision of an antigen made from embryonicconnective tissue cells, which are obtained from the substance known asWhartons jelly, found in the human umbilical cord, and containingembryonic connective tissue, particularly stellate connective tissuecells, treating and mixing the cells to obtain a protein, prepared andtreated in accordance with a predetermined sequence and formula.

An additional object is to provide a highly perfected method ofsynthesis for an antigen whereby a pure antigen is obtained which in useis characterized by a high degree of accuracy.

A further object is to provide a new antigen for determining whether ornot a human being is afilctecl with sarcoma by injecting the antigenintradermally.

A still further object of the invention and discovery is to provide atest to determine whether or not a human being is afilicted with sarcomaby giving intradermal injections of a suitable antigen and preservingthe results of the injections particularly with regard to the formationor absence of formation of pseudopods, the occurrence of a pseudopoclicreaction indicating positively the presence of specific sarcomatousprotein in the body of the patient and, accordingly, the presence of asarcomatous growth in the individual.

Numerous other objects and advantages will be apparent throughout theprogress of the following specification.

Proceeding on the basis that sarcoma cells are embryonic connectivetissue cells, always remain embryonic and never mature, a protein from55 these cells, when brought into contact with a patients serumcontaining a characteristic sarcomatous protein causes a flocculation orprecipitation or a reaction.

The embryonic connective tissue cells from which the antigen is made arepreferably obtained from the gelatinous substance present in theumbilical cord, and particularly the human umbilical cord whichsubstance is technically known as Wharton's jelly.

Human umbilical cords or the umbilical cords of mammalians are collectedin a sterile flask containing a sterile saline solution made up in thepreparation of 8.5 grams of sodium chloride in a liter of distilledwater. The cords should be not more than one day old when used, and anycords which are not white and in good condition should be discarded. Thecords'are cleaned of any excess blood, in the three vessels of the cord,by pressing out the cord with a clean gauze sponze. The cleaning of thecord is facilitated if the cord is cut into short pieces of about fourinches in length. The cord should be kept either in or constantly wettedwith saline solution while the pieces of the cord are being cleaned,which pieces may be cleaned on several thicknesses of sterile papertowels. After the blood has been squeezed out of the cord, a surgicalknife is employed to completely cut-around the large vessel and it iscarefully dissected from its place. The large vessel of the cord is cutopen and wiped free from blood with a clean gauze sponge which may bewet with saline solution. The two smaller vessels,'of the cord aresimilarly dissected from their normal position with a knife and usingtoothed forceps. The clean pieces of the cords are collected insterilized saline solution until the cleaning process has beencompleted. The clean pieces of the cords are then put into a meat pressof the cone-type, and ground, collecting the resultant jelly in acontainer separate from the pieces of the cord. The pieces of the cordmay be put through the press several times in order to remove all thejelly therefrom. Any small pieces of the cord which may get into thejelly should be immediately removed. This may be done by filtering thejelly through a sterilized Goocl'i crucible padded with several layersof surgical gauze, or filtering through glass wool. The jelly which isso obtained is termed Wharton's jelly. The Wharton's jelly is thencarefully and slowly dried in an oven until it has dried sufficiently,so that it may be ground into powder. Care should be taken that thejelly will not become scorched during the drying operation.

The dried cells are next treated to a process of washing or extractingwith ether in the following manner.

The cells, dried in the above manner, are then placed in a test tube andether, 1. e., ethyl ether known commercially as sulfuric ether, is addedto cover' the cells preferably in three times the approximate volume ofthe cells. The mixture is shaken to completely wash the cells thereinand is permitted to stand for two hours. The container is carefullycovered meanwhile. After this period the ether may be poured off. Ifsuflicient settling has not taken place, the product should becentrifuged at a medium speed for five minutes or so when the ether maybe readily decanted from the mass of cells. After this operation it ispreferred that the mass of cells, still wet with ether, be permitted todry. This is, of course, readily accomplished in view-of the volatilenature of ether. Fresh ether is again added in about the same amount asbefore and the operation again carried out in the same manner. Thisoperation is repeated until thecells have been washed in ether for threeseparate times.

When the jelly is dried it is placed in acetone of five times itsvolume. The acetone is then poured off from the dried jelly, and whenall the acetone has completely evaporated from the jelly, the driedjelly is placed in a mortar and ground'to an impalpable powder, to aboutthe consistency of a fine talcum powder. The dried powdered jelly soobtained should be stored in sterilized airtight containers.

The purpose of the oven drying operation is for dehydration. Thedehydration of the cells can be done better, and quicker, and withoutdanger of scorching. by dehydrating the jelly with acetone only.

The dried powdered jelly of the umbilical cord is then extracted withone-tenth normal sodium hydroxide in the proportion of one-tenth gram totwo-tenths gram, preferably tw'o-tenths gram, of dried cells to tencubic centimeters of the onetenth normal sodium hydroxide. The normalsodium hydroxide (NaOH, C. P.) is made up in the proportions of oneliter of distilled Water in a volumetric flask to four grams of sodiumhydroxide, C. P. The powdered cells to which the one-tenth normal sodiumhydroxide is added is first ground to a smooth paste with a few cubiccentimeters of the sodium hydroxide, after which time the remainingamount of the ten cubic centimeters of the normal sodium hydroxide isadded slowly. The cells and sodium hydroxide are carefully mixed so thata perfectly smooth suspension of the cells in the sodium hydroxide willbe obtained. The extract, the cells and the sodium hydroxide, are thenpoured into large containers such as large test tubes and allowed tostand for 24 hours. After thistime it is preferred that the liquid andits contents be centrifuged at medium speed for twenty minutes in orderto permit complete and full separation of the supernatant liquid, whichmay then be pipetted off or otherwise separated. The volume of thesupernatant solution should be measured with a sterilized graduate. Thissupernatant solution is the alkaline extract of the jelly, and is placedin a sterile bottle of a volume more than twice that of the extracts soas to allow for the addition of an acid and buffer solution which isused in neutralizing.

The acid and buffer solution for neutralizing the alkaline extract ismade up in the proportion of 2.27 of anhydrous C. P. primary potassiumphosphate (KH2PO4) and 4.235 cubic centimeters of concentratedhydrochloric acid (HCI), specific gravity 1.18-1.19, per cent. solutionmade up to one liter with distilled water in a volumetric flask. Thisgives a solution which is .05 normal with respect to the hydrochloricacid and .05 normal with respect to primary potassium phosphate,anhydrous.

An equal amount of the acid and buffer solution just described may beadded to the alkaline extract which has been pipetted off from the cellsand measured. The acid solution should be added slowly, and thesolutions carefully stirred and gently agitated while the acid andbuffer solution is being added. After an equal amount of the acid andbuffer solution has been added to the alkaline extract of the cells, afew more cubic centimeters of the acid and buffer solution may be added.The resultant solution should then be tested to see if theneutralization is nearing the end point. This testing should be repeatedfrequently to make certain that the titration does not go past the endpoiht, which is pH 6.9 for this process. too acid, the protein of theantigen'will be precipitated. The antigen should be checkedelectrometrically if possible, and if not possible, it

may be checked by the use of a spot plate, and checked against astandard solution of pH 6.9.

Brom-thymol-blue is used as an indicator.

The standard solution just referred to, is well known, and is made up ofanhydrous primary potassium sulphate, molar, 9.078 grams of of 4.9 partsof the solution of primary potassiumphosphate to 5.1 parts of secondarysodium phosphate solution. The mixture of these two solutions gives asolution with a pH of 6.9.

The solution is now passed through a number V Berkfield filter to removeany foreign matter or bacterial contamination. This filtering step maybe applied prior to the addition of acid and buffer in another preferredembodiment of the invention, that is after decantation or pipetting offthe supernatant'alkaline extract.

When the end point of the titaration is reached, as ascertained by theelectrometric method, or by the matching of the antigen to the standardsolution of pH 6.9, a preservative consisting of a solution oftrY-cresol and glycerin C. P. in the proportion of one part oftri-cresol to two parts of glycerin, may be added.

The total volume of the finished antigen, that is, including the volumeof the alkaline extract and the added volume of the acid and buffersolution, is calculated, and to each ten cubic centimeters of antigenthere is added two drops of the tri-cresol and glycerin preservative.Two drops are added by the use of a capillary pipette having an internaldiameter of one millimeter at the dropping end. A sterile stopper isplaced in the bottle containing the antigen, and its preservative, andthe solution should be shaken thoroughly so that the preservative willbe disbursed evenly throughout the antigen. A rubber stopper of the captype'is preferably used on the bottle containing the antigen, so thatthe solution may' be withdrawn by means of a syringe and'needle forconducting the intradermal test, and the bottle need not be opened.

After the preservative has been added to the antigen, and the. solutionthoroughly shaken, the finished antigen is heated to about C. for fiveminutes. This treatment apparently has the function of destroying orchanging to a harmless form any impurities which might interfere withthe use of the antigen. The antigen may be withdrawn from the containerby means of a Pyrex syringe, and put into small vials for use. Vials offive cubic centimeter capacity have been found convenient.

It is essential to use glass containers of Pyrex for thereason thatordinary glass seems to give up alkali to the product and thus changeits character.

Fivephundredths (.05) of one (1) c. c. of the above antigen is drawn oflin a small syringe to which there is attached a very fine short needle.The antigen is injected intradermally, after first sterilizing and treating the surface of the pa- Ifthe resultant solution becomesunitsskinand rendering it perfectly dry. The

injection is performed by-stretching the skin with one hand andinjecting the antigen intradermally, the injection beingmade by theusual intradermal method. In positive cases, that'is, in

cases where the patient examined has sarcoma,

a slight area of inflammation will be noticed surrounding the smallbubble termed a bleb, which occurs from the injection, andpseudopodswill form, pseudopods being radial elongations extendingoutwardly from the edges of the bleb.'

In negative cases, that is, in cases where the patient does not havesarcoma, no pseudopod formation will take place.

The above described antigen is for the intradermal test, and is known asthe dry method. A similar method known as the wet method is the same asthe present method, except the Whartons jelly is not dried, being leftin its wet state.

The invention and discovery herein set forth' designates, to a highdegree of certainty, whether or not a patient is afflicted with sarcoma.The

antigen herein described are made from embryonic connective tissue cellsof the umbilical cord, and while the exact methods and tests hereindescribed are preferable, it is to be understoodthat various changes, toa certain degree,

may be made, without departing from the spirit of the invention orsacrificing any of its advantages, and the right is hereby reserved tomake all such changes as fairly fall within the scope of the followingclaims.

The invention is hereby claimed as follows:

1. An antigen specific to the diagnosis of sarcoma by intradermalinjection comprising a neutralized, inorganic alkaline hydroxide extractof embryonic connective tissue cells obtained from embryo" mammals.

2. An antigen specific to the diagnosis of sarcoma by intradermalinjection comprising a neu-' tralized, inorganic, alkaline hydroxideextract of embryonic connective tissue cells obtained from embryomammals having a pH of substantially 6.9.

3. An antigen specific to the diagnosis of sarcoma by intradermalinjection comprising a neutralized, sodium hydroxide extract ofembryonic connective tissue cells from embryo mammals.

4. The process of making an antigen for in tradermal use to determinediagnostically the existence of sarcoma comprising extracting embryonicconnective tissue cells obtained from embryo mammals with an inorganic,alkaline hydroxide, separating the extract, and then neuexistence ofsarcoma in a mammalian comprising an extract of embryonic connectivetissue cells obtained from embryo mammals and adapted for intradermalinjection, which extract contains a specific protein homologous to thespecific sarc'omatous protein of a mammalian having sarcoma and whichproduces a skin reaction by pseudopod-formation when the antigen isinjected intradermaliy into an animal affiicted with sarcoma.

'7. An antigen specific to the diagnostic determination of sarcoma byintradermal injection comprlslng a neutralized, sodium hydroxide ex-Vtract 01' embryonic connective tissue cells obtained from the humanembryo.

8. The process of making an antigen for intradermal use to determinediagnostically the existence of sarcoma which comprises extractingembryonic connective tissue cells obtained from Wharton's jelly withsodium hydroxide. separating the extract, and then neutraliflng andbuffer- 1, ing the extract.

9. The process of making an antigen for intradermal use to determinediagnostically the existence of sarcoma comprising extracting embryonicconnective tissue cells from the human umbilical cord with substantiallyone-tenth normal sodium hydroxide, and then adding an acid and buflersolution to neutralize the extract to a pH of substantially 6.9.

BENJAMIN aausxm.

